It is proposed to continue studies of protein metabolism in Bacillus subtilis in four major areas. (1) Homogeneous preparations of the intracellular metalloserine protease (ISP) will be used to prepare rabbit antisera. The antibodies recovered will be labeled with ferritin and used to attempt to localize this protease to a certain region of the cell (e.g., cytoplasm, mesosome, or cytoplasmic membrane). (2) Antibodies against ISP and bacillopeptidase F (previously prepared) will be used to determine similarities, if any, between these two activities. (3) Attempts will be made to isolate mutants which are deficient in the protease (uncharacterized) which we have inferred to be present in log phase cells. (4) Methods previously developed to analyze B. subtilis proteins on 2-dimensional O'Farrell gels will be extended. Using 3H- and 14C-leucine labeling techniques we will attempt to quantitatively determine protein turnover of individual proteins by densitometric analysis of X-ray films exposed to slab gels containing labeled proteins.